Activatable recombinant neurotoxins

ABSTRACT

Compositions comprising activatable recombinant neurotoxins and polypeptides derived therefrom. The invention also comprises nucleic acids encoding such polypeptides, and methods of making such polypeptides and nucleic acids.

This application is a continuation and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 11/326,265, now U.S.Pat. No. 7,419,676 filed Jan. 5, 2006, a divisional that claims prioritypursuant to 35 U.S.C. §120 to U.S. patent application Ser. No.09/648,692, filed Aug. 25, 2000, now U.S. Pat. No. 7,132,259, anapplication that claims priority pursuant to 35 U.S.C. §119(e) toprovisional application Ser. No. 60/150,710, filed Aug. 25, 1999, eachof which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention concerns methods and compositions useful in the fields ofneurobiology, molecular biology, and medicine, as well as methods forthe production of potentially toxic therapeutic agents and derivativesthereof. The invention also concerns recombinant clostridial neurotoxins(particular botulinum neurotoxins), modified versions thereof, andmethods of making such molecules, for use as therapeutic agents,transporter molecules, adducts, and the like.

BACKGROUND OF THE INVENTION

Neurotoxins, such as those obtained from Clostridium botulinum andClostridium tetani, are highly potent and specific poisons of neuralcells, and other cells when delivered within such cells for therapeuticpurposes. These Gram positive bacteria express two related but distincttoxins types, each comprising two disulfide-linked amino acid chains: alight chain (L) of about 50 KDa and a heavy chain (H) of about 100 KDa,which are wholly responsible for the symptoms of these diseases. Theholotoxin is synthesised in vivo as a single chain, then nicked in apost-translational modification to form the active neurotoxin comprisingthe separate L and H chains.

The tetanus and botulinum toxins are among the most lethal substancesknown to man, having a lethal dose in humans of between 0.1 ng and 1 ngper kilogram of body weight. Tonello et al., Adv. Exp. Med. & Biol.389:251-260 (1996). Both toxins function by inhibiting neurotransmitterrelease in affected neurons. The tetanus neurotoxin (TeTx) acts mainlyin the central nervous system, while botulinum neurotoxin (BoNT) acts atthe neuromuscular junction and other cholinergic synapses in theperipheral nervous system; both act by inhibiting neurotransmitterrelease from the axon of the affected neuron into the synapse, resultingin paralysis.

The tetanus neurotoxin (TeTx) is known to exist in one immunologicallydistinct type; the botulinum neurotoxins (BoNT) are known to occur inseven different immunogenic types, termed BoNT/A through BoNT/G. Whileall of these types are produced by isolates of C. botulinum, two otherspecies, C. baratii and C. butyricum also produce toxins similar to /Fand /E, respectively. See e.g., Coffield et al., The Site and Mechanismof Action of Botulinum Neurotoxin in Therapy with Botulinum Toxin 3-13(Jankovic J. & Hallett M. eds. 1994), the disclosure of which isincorporated herein by reference.

Regardless of type, the molecular mechanism of intoxication appears tobe similar. In the first step of the process, the toxin binds to thepresynaptic membrane of the target neuron through a specific interactionbetween the heavy (H) chain and a cell surface receptor; the receptor isthought to be different for each type of botulinum toxin and for TeTx.Dolly et al., Seminars in Neuroscience 6:149-158 (1994), incorporated byreference herein. The carboxyl terminus of the heavy chain appears to beimportant for targeting of the toxin to the cell surface. Id.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This last step is thought to be mediated by the amino terminus ofthe H chain, which triggers a conformational change of the toxin inresponse to a pH of about 5.5 or lower. Endosomes are known to possess aproton pump which decreases intra endosomal pH. The conformational shiftexposes hydrophobic residues in the toxin, which permits the toxin toembed itself in the endosomal membrane. The toxin then translocatesthrough the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the H and light (L)chain. The entire toxic activity of botulinum and tetanus toxins iscontained in the L chain of the holotoxin; the L chain is a zinc (Zn⁺⁺)endopeptidase which selectively cleaves proteins essential forrecognition and docking of neurotransmitter-containing vesicles with thecytoplasmic surface of the plasma membrane, and fusion of the vesicleswith the plasma membrane. TeTx, BoNT/B BoNT/D, BoNT/F, and BoNT/G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the cytosolicdomain of VAMP extending from the surface of the synaptic vesicle isremoved as a result of any one of these cleavage events. Each toxin(except TeTx and BoNT/B) specifically cleaves a different bond.

BoNT/A and /E selectively cleave the plasma membrane-associated proteinSNAP-25; this protein, which is also cleaved by BoNT/C1 (Foran et al.,Biochem. 35:2630-2636 (1996)), is predominantly bound to and present onthe cytosolic surface of the plasma membrane. BoNT/C cleaves syntaxin,an integral protein having most of its mass exposed to the cytosol.Syntaxin interacts with the calcium channels at presynaptic terminalactive zones. See Tonello et al., Tetanus and Botulinum Neurotoxins inIntracellular Protein Catabolism 251-260 (Suzuki K. & Bond J. eds.1996), the disclosure of which is incorporated by reference as part ofthis specification.

Both TeTx and BoNT are taken up at the neuromuscular junction. BoNTremains within peripheral neurons, and blocks release of theneurotransmitter acetylcholine from these cells. Through its receptor,TeTx enters vesicles that move in a retrograde manner along the axon tothe soma, and is discharged into the intersynaptic space between motorneurons and the inhibitory neurons of the spinal cord. At this point,TeTx binds receptors of the inhibitory neurons, is again internalized,and the light chain enters the cytosol to block the release of theinhibitory neurotransmitters 4-aminobutyric acid(GABA) and glycine fromthese cells.

Because of its specifically localized effects, minute doses of BoNT havebeen used since 1981 as therapeutic agents in the treatment of patientssuffering from dystonias, including strabismus (misalignment of theeye), bephlarospasm (involuntary eyelid closure) and hemifacial spasm.See e.g., Borodic et al, Pharmacology and Histology of the TherapeuticApplication of Botulinum Toxin in Therapy with Botulinum Toxin 119-157(Jankovic J. & Hallett eds. 1994), hereby incorporated by referenceherein. Of the seven toxin types, BoNT/A is the most potent of theBoNTs, and the best characterized. Intramuscular injection of spastictissue with small quantities of BoNT/A has also been used effectively totreat spasticity due to brain injury, spinal cord injury, stroke,multiple sclerosis and cerebral palsy. The extent of paralysis dependson both the dose and volume delivered to the target site.

Although the L chain is the moiety responsible for neural intoxication,it must be delivered to the neural cytoplasm in order to be toxic.Similarly, the single chain holotoxin pro-forms exhibit relatively lowtoxicity until they are cleaved at one or more peptide bonds in anexposed loop region between their H and L chains to create thefully-active mature neurotoxins. As implied in the mechanism providedabove, the H chain of each neurotoxin is essential for cell receptorbinding and endocytosis, while both the L and the H chains (and anintact disufide bond) are required for translocation of the toxin intothe cytoplasm. As indicated above, the L chain alone is responsible forthe toxicity caused by inhibition of acetylcholine secretion.

Despite the clear therapeutic efficacy of clostridial neurotoxinpreparations, industrial production of the toxin is difficult.Production of neurotoxin from anaerobic Clostridium cultures is acumbersome and time-consuming process including a multi-steppurification protocol involving several protein precipitation steps andeither prolonged and repeated crystallisation of the toxin or severalstages of column chromatography. Significantly, the high toxicity of theproduct dictates that the procedure must be performed under strictcontainment (BL-3). During the fermentation process, the foldedsingle-chain neurotoxins are activated by endogenous clostridialproteases through a process termed nicking. This involves the removal ofapproximately 10 amino acid residues from the single-chain to create thedichain form in which the two chains remain covalently linked throughthe interchain disulfide bond.

The nicked neurotoxin is much more active than the unnicked form. Theamount and precise location of nicking varies with the serotypes of thebacteria producing the toxin. The differences in single-chain neurotoxinactivation and, hence, the yield of nicked toxin, are due to variationsin the type and amounts of proteolytic activity produced by a givenstrain. For example, greater than 99% of C. botulinum type Asingle-chain neurotoxin is activated by the Hall A C. botulinum strain,whereas type B and E strains produce toxins with lower amounts ofactivation (0 to 75% depending upon the fermentation time). Thus, thehigh toxicity of the mature neurotoxin plays a major part in thecommercial manufacture of neurotoxins as therapeutic agents.

The degree of activation of engineered clostridial toxins is, therefore,an important consideration for manufacture of these materials. It wouldbe a major advantage if neurotoxins such as BoNT and TeTx could beexpressed in high yield in rapidly-growing bacteria (such asheterologous E. coli cells) as relatively non-toxic single-chains (orsingle chains having reduced toxic activity) which are safe, easy toisolate and simple to convert to the fully-active form.

With safety being a prime concern, previous work has concentrated on theexpression in E. coli and purification of individual H and L chains ofTeTx and BoNT; these isolated chains are, by themselves, non-toxic; seeLi et al., Biochemistry 33:7014-7020 (1994); Zhou et al., Biochemistry34:15175-15181 (1995), hereby incorporated by reference herein.Following the separate production of these peptide chains and understrictly controlled conditions the H and L subunits can be combined byoxidative disulphide linkage to form the neuroparalytic di-chains.Unfortunately, this strategy has several drawbacks.

Firstly, it is not practical to express and isolate large amounts of theindividual chains; in particular, in the absence of the L chain theisolated H chain is quite insoluble in aqueous solution and is highlysusceptible to proteolytic degradation. Secondly, the in vitro oxidationof the individually expressed and purified H and L chains to produce theactive di-chain is very inefficient, and leads to low yields of activetoxin and the production of many inactive incorrectly folded or oxidizedforms. The purification of the correctly folded and oxidized H and Lchain-containing toxin is difficult, as is its separation from theseinactive forms and the unreacted separate H and L chains.

It would therefore be useful and advantageous to express clostridialneurotoxins as inactive (or less active) single-chain forms, toeliminate the need for the time-consuming and inefficient reconstitutionof the constituent chains, to maintain solubility of the protein chains,to reduce protein misfolding and consequent susceptibility to proteaseattack, to improve toxin yield, and/or to provide a simple method forthe purification of the toxin.

Additionally, it would be useful to engineer these toxins to providesingle-chain, modified neurotoxin molecules having novel therapeuticproperties and/or longer duration of action, or toxic or non-toxic formsfor use as transport molecules capable of delivering a therapeuticmoiety to nerve or other cell types. By expressing such proteins as asingle chain, the yield and purification of the engineered proteinswould be vastly improved.

SUMMARY OF THE INVENTION

The present invention is directed to recombinant and isolated proteinscomprising a functional binding domain, translocation domain, andtherapeutic domain in which such proteins also include an amino acidsequence that is susceptible to specific cleavage in vitro followingexpression as a single chain. Such proteins may include clostridialneurotoxins and derivatives thereof, such as those proteins disclosed inU.S. Pat. No. 5,989,545 and International Patent Application WO95/32738,both incorporated by reference herein.

In one embodiment of the invention the protein comprises the functionaldomains of a clostridial neurotoxin H chain and some or all of thefunctions of a clostridial neurotoxin L chain in a single polypeptidechain, and having an inserted proteolytic cleavage site located betweenthe H domain and the L domain by which the single chain protein may becleaved to produce the individual chains, preferably covalently linkedby a disulfide linkage. The invention also includes methods of makingsuch proteins and expressing them within a cell, as well as nucleic acidvectors for the transfer and expression of the nucleotide sequenceregions encoding such proteins and cells containing such vectors. Theproteolytic cleavage sites comprise amino acid sequences that areselectively recognized and cleaved by a specific enzyme.

In a preferred aspect of the invention, the expressed single-chainproteins comprise the biologically active domains of the H chain and Lchain of a clostridial neurotoxin. Scission at the internal proteolyticcleavage site separating the chain domains thus results in theactivation of a neurotoxin having full activity.

In another aspect of the invention the single-chain proteins comprise abinding domain targeted to a cell receptor other than one borne by amotor neuron. Such a binding domain may specific bind to, for example, asensory afferent neuron, or to a non-neuronal cell type or tissue, suchas pancreatic acinar cells. The single-chain proteins will contain atranslocation domain similar to that of clostridial neurotoxins, and atherapeutic moiety. The therapeutic moiety may be a clostridialneurotoxin light chain, or may be a different therapeutic moiety such asan enzyme, a transcribable nucleotide sequence, growth factor, anantisense nucleotide sequence and the like.

Preferably, the toxins and toxin-based proteins of the present inventionwill be tailored to contain an additional amino acid sequence comprisinga binding tag able to bind a target compound at sufficiently highefficiency to facilitate rapid isolation of the toxin protein. Proteinscontaining such binding sites are many and well known to those of skillin the art, and may comprise, without limitation, monoclonal antibodies,maltose binding protein, glutathione-S-transferase, protein A, a His₆tag, and the like.

Because such proteins exhibit binding selectivity to a certain compoundor compound type, the target compound may be immobilized to a solidsupport, including without limitation, a chromatography resin ormicrotiter well and used for affinity purification of the modifiedtoxin. The toxin molecule can then be eluted by standard methods, suchas through the use of a high salt solution or specific antagonist.

To minimize the safety risk associated with handling neurotoxin, thetoxins of the this aspect of the present invention are expressed astheir low activity (or inactive) single-chain proforms, then, by acarefully controlled proteolytic reaction in vitro, they are activated,preferably to the same potency level as the native neurotoxin from whichthey were derived. To improve the efficiency and rate of proteolyticcleavage the engineered proteolytic cleavage sites can be designed tooccur in a specially-designed loop between the H and L portions of thesingle amino acid chain that promotes accessibility of the protease tothe holotoxin substrate.

To reduce the risk of unintentional activation of the toxin by human orcommonly encountered proteases, the amino acid sequences of the cleavagesite are preferably designed to have a high degree of specificity toproteolytic enzymes which do not normally occur in humans (as eitherhuman proteases or occurring in part of the foreseeable human fauna andflora). A non-exclusive list of examples of such proteases includesbovine enterokinase, which cleaves the amino acid sequence DDDDK (SEQ IDNO: 15); tobacco etch virus (TEV) protease, which cleaves the sequencesEXXYXQS (SEQ ID NO: 22) and EXXYXQG (SEQ ID NO: 23); GENENASE® fromBacillus amyliquifaciens, which cleaves the sequence HY or YH; andPRESCISSION® protease from human rhinovirus 3C, which cleaves the aminoacid sequence LEVLFQGP (SEQ ID NO: 16). As used above, the letter Xindicates any amino acid. All amino acid sequences shown in the presentspecification are in the direction from amino terminus to carboxylterminus, and all nucleotide sequences from 5′ to 3′, (from left toright) unless otherwise indicated.

In an aspect of the invention the single-chain polypeptide is anisolated polypeptide. By “isolated” is meant removed from its naturalenvironment. For example, for a protein expressed within the cell,isolation includes preparation of a cell lysate as well as subsequentpurification steps. A protein expressed extracellularly may be isolatedby, for example, separation of the supernatant from the cells as well asany subsequent purification steps.

In another aspect of the invention the interchain loop region of the C.botulinum subtype E neurotoxin, which is normally resistant toproteolytic nicking in the bacterium and mammals, is modified to includethe inserted proteolytic cleavage site, and this loop region used as theinterchain loop region in the single-chain toxin or modified toxinmolecules of the present invention. It is believed that using the loopfrom C. botulinum subtype E will stabilize the unnicked toxin moleculein vivo, making it resistant to undesired cleavage until activatedthrough the use of the selected protease.

In yet another aspect of the invention are contemplated compositionscomprising recombinant forms of BoNT/E expressed as a single chainpolypeptide.

In still another aspect are contemplated recombinant chimeric and/ormodified toxin derivatives expressed as a single chain polypeptide. Suchpolypeptide may be molecular transporters, such as, without limitation,those disclosed in Dolly et al., European Patent Specification EP 0 760681 B1, incorporated by reference herein.

In a further aspect the invention includes neurotoxin derivativescomprising at least a portion of a light chain from one clostridialneurotoxin or subtype thereof, and at least a portion of a heavy chainfrom another neurotoxin or neurotoxin subtype, as well as methods fortheir production. In one embodiment the hybrid neurotoxin may containthe entire light chain of a light chain from one neurotoxin subtype andthe heavy chain from another neurotoxin subtype. In another embodiment,a chimeric neurotoxin derivative may contain a portion (e.g., thebinding domain) of the heavy chain of one neurotoxin subtype, withanother portion of the heavy chain being from another neurotoxinsubtype. Similarly or alternatively, the therapeutic element maycomprise light chain portions from different neurotoxins.

Such hybrid or chimeric neurotoxin derivatives are useful, for example,as a means of delivering the therapeutic benefits of such neurotoxins topatients who are immunologically resistant to a given neurotoxinsubtype, to patients who may have a lower than average concentration ofreceptors to a given neurotoxin heavy chain binding moiety, or topatients who may have a protease-resistant variant of the membrane orvesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin). Creation ofrecombinant chimeric or hybrid neurotoxin derivatives having a lightchain with different substrate would permit such patients to respond toneurotoxin therapy.

With regard to immunological resistance, it is known that mostneurotoxin epitopes exist on the heavy chain portion of the toxin. Thusif a patient has neutralizing antibodies to, for example BoNT/A, achimeric neurotoxin containing the heavy chain from BoNT/E and the lightchain from BoNT/A (which has a longer duration of therapeutic activitythan other neurotoxin light chains) would overcome this resistance.Likewise if the patient has few cell surface receptors for BoNT/A, thechance are great that the same patient would have adequate receptors toanother BoNT subtype. By creating a hybrid or chimeric neurotoxin (suchas one containing at least a portion of a heavy chain selected from thegroup consisting of HC_(A), HC_(B), HC_(C1), HC_(D), HC_(E), HC_(F), andHC_(G) and a at least a portion of a light chain selected from adifferent clostridial neurotoxin subtype, said light chain beingselected from the group consisting of LC_(A), LC_(B), LC_(C1), LC_(D),LC_(E), LC_(F), and LC_(G)) combining the heavy chain of that subtypewith the most therapeutically appropriate light chain (for example, theBoNT/A light chain) the patient could better respond to neurotoxintherapy.

Another advantage of the hybrid or chimeric neurotoxin derivativesdescribed above is related to the fact that certain of the light chains(e.g., LC_(A)) have a long duration of action, others having a shortduration of action (e.g., LC_(E) AND LC_(F)) while still others have anintermediate duration of activity (e.g., LC_(B)). Thus, hybrid andchimeric neurotoxins represent second and third generation neurotoxindrugs in which the neurotoxin activity may be tailored to a specifictherapeutic need or condition, with different drugs having differentactivities, substrate specificities or duration of activity.

Such hybrid or chimeric neurotoxins would also be useful in treating apatient (such as a soldier or laboratory worker) who has been inoculatedwith the pentavalent BoNT vaccine. Such vaccines do not contain BoNT/F;thus, combining the appropriate light chain with the BoNT/F heavy chainwould create a therapeutic agent which is effective in such a patientwhere current therapeutic neurotoxins may not be.

The same strategy may be useful in using derivatives of clostridialneurotoxins with a therapeutic moiety other than an active neurotoxinlight chain. As the heavy chain of such an agent would be derived from aneurotoxin, it may be advantageous to use a lesser known, or rarer heavychain to avoid resistance mechanisms neutralizing the effectiveness ofthe therapeutic neurotoxin derivative.

By the same token, the binding moiety may be one other than a bindingmoiety derived from a clostridial neurotoxin heavy chain, thus providinga targeting function to cell types other than motor neurons.

Also included herein are methods for the construction, expression, andpurification of such molecules in high yield as biologically activeentities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagrammatic view of the single-chain TeTx construct inplasmid pTrcHisA and the nucleotide sequence of the junction region.

FIG. 1B shows the and amino acid sequence connecting the carboxylterminus of the L chain and the amino terminus of the H chain and anengineered loop region containing an enterokinase cleavage site.

FIG. 2A is a representation of a Western blot of an SDS-PAGE gel of cellextracts of E. coli JM 109 transformants containing 2 differentrecombinant single-chain toxins, either before or after induction ofplasmid protein expression with IPTG. The antibody used for detection isan anti-His₆ monoclonal antibody.

FIG. 2B is a Western blot of IPTG-induced cell extracts from cellstransformed with the E234A construct.

FIG. 3A shows the results of an experiment in which affinity purifiedrecombinant single-chain (SC) TeTx is nicked with enterokinase, thenseparated using SDS-PAGE and visualized using Commassie Brilliant Blueunder reducing and non-reducing conditions.

FIG. 3B shows the results of an experiment in which affinity purifiedrecombinant single-chain (SC) TeTx is nicked with enterokinase, thenseparated using SDS-PAGE under reducing and non-reducing conditions andsubjected to a Western blot using anti TeTx heavy chain antibody.

FIG. 4 is a plot of the degree of paralysis induced in a nerve/musclepreparation in vitro using native TeTx, and recombinant single chainneurotoxin before, and after nicking as a function of time.

FIG. 5 is a depiction of the peptide fragments generated upon incubationof the recombinant single-chain TeTx with trypsin and Arg C protease,and deduction, from the N-terminal sequences of one of the resultingfragments, of the amino acid sequence recognized by these agents.

FIG. 6 shows the digestion of unnicked SC WT TeTx and SC R496G TeTx withvarious concentrations of trypsin.

FIG. 7 shows the inhibitory effect upon TeTx stimulated inhibition ofCa⁺⁺-dependent neurotransmitter release of preincubating cerebellarcells with the E234A mutant TeTx. Pre-incubation of cerebellar neuronswith nicked (FIG. 7A) or unnicked (FIG. 7B) TeTx E234A at 4° C. followedby the addition of 0.2 nM native toxin, antagonized the native toxin'sinhibition of transmitter release at 37° C. to similar extents.

FIG. 8 shows the effect upon Ca⁺⁺-dependent neurotransmitter release ofcerebellar neurons upon exposure to native, recombinant E234A mutantsingle-chain, and the recombinant R496G mutant single chain TeTx.

FIG. 9 shows the inhibitory effect upon TeTx-stimulated paralyticactivity of preincubating mouse hemi diaphrams with the E234A mutantTeTx.

FIG. 10 shows the scheme for construction of a plasmid encodingsingle-chain BoNT/E, and an agarose gel electrophoretogram of the PCRfragment obtained during the construction of the plasmid.

FIG. 11 shows the scheme for construction of a plasmid encoding theE212Q proteolytically inactive single-chain BoNT/E mutant, and anagarose gel electrophoretogram of the inverse PCR fragment obtainedduring the construction of the plasmid.

FIG. 12 shows the expression and purification scheme for recombinantsingle-chain BoNT/E, and a SDS-PAGE electrophoretogram and Western blotof the purification fractions.

FIG. 13 shows SDS-PAGE electrophoretograms under reducing andnon-reducing conditions of native recombinant unnicked, and recombinantnicked BoNT/E, and Western Blots directed towards the heavy and lightchains of the toxin.

FIG. 14 shows the results of incubating native BoNT/E, recombinantnicked and un-nicked BoNT/E, and the E212Q mutant with aGST-SNAP-25-[140-205] protease substrate.

FIG. 15 shows the effect upon Ca⁺⁺-dependent glutamate release ofincubating cerebellar cells with native BoNT/E, un-nicked recombinantsingle chain BoNT/E, and nicked recombinant single chain BoNT/E.

FIG. 16A shows the effects on muscle tension of incubating mousephrenic-nerve hemi-diaphragms with 0.2 nM recombinant nicked BoNT/E (

) or 0.2 nM native BoNT/E (□).

FIG. 16B shows the effects on muscle tension of incubating mousephrenic-nerve hemi-diaphragms with 1 nM recombinant un-nicked (

), 1 nM recombinant nicked (●) or 0.05 nM recombinant nicked (∇) BoNT/E.

FIG. 17 shows the attenuation of paralytic activity on mousephrenic-nerve hemi-diaphragms of preincubation with the inactive E212Qmutant prior to exposure to native nicked BoNT/E toxin.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods of the present invention involve modifiedneurotoxins, their synthesis and use. Di-chain neurotoxins that arenormally activated by scission of a single chain polypeptide byindigenous proteases can be modified at the nucleic acid level byalteration or removal of the nucleotide sequence encoding the indigenousprotease cleavage site and insertion of a nucleotide sequence encodinganother different proteolytic cleavage site resistant to cleavage byhost cell or human proteases. The inserted amino acid sequence isdesigned to be cleaved in vitro through the use of a cleaving agentchosen in advance of expression that is absent from both human and hostcell tissue.

The inserted amino acid sequence may be chosen to confer susceptibilityto a chemical agent capable of cleaving peptide bonds, such as cyanogenbromide. However, and much more preferably, the encoded amino acidsequence may comprise a proteolytic cleavage site highly specific for aselected protease. The selected protease is one not normally present inthe cell expressing the single chain toxin molecule (for example, E.coli or the native clostridial bacterium).

In another aspect, the invention is drawn to recombinant single-chainmodified clostridial neurotoxins that may be cleaved at will by aprotease to provide an active di-chain molecule. Such modifiedneurotoxins need not be toxic; in certain of these proteins theenzymatic activity of the toxin L chain may be abrogated, and the toxinjoined to a drug or other bioactive agent having therapeutic activity.Alternatively, in certain other modified neurotoxins the L chain isenzymatically active, but portions of the H chain are modified toprovide specificity to target cells other than the natural target of theneurotoxin, while maintaining the translocation andendocytosis-stimulating activities of the native toxin.

Modified neurotoxins such as those described in this aspect of theinvention are disclosed in, for example, International PatentPublications WO95/32738, WO 99/55359, WO96/33273, WO98/07864 andWO99/17806, these publications are incorporated by reference herein. Thepresent invention provides single-chain, cleavable versions of thesemolecules and improved methods of making such molecules.

In another aspect, the invention comprises a modified clostridialneurotoxin derived from tetanus toxin (TeTx), or one or more of thebotulinum toxin (BoNT) subtypes in which the naturally-occurringinterchain loop region has been replace with a modified loop regioncomprising a different amino acid sequence conferring 1) resistance tocleavage by host proteases or autolytic action, and/or 2) lability to aselected protease. Preferably the cleavage site is highly specific forthe selected protease.

The interchain loop region of certain clostridial neurotoxins, forexample, BoNT/E, is naturally resistant to proteolytic cleavage in vivo.This protease resistance may reflect a secondary or tertiary structurethat makes the loop more resistant to indigenous proteases than otherclostridial neurotoxins. In one embodiment of the present invention,therefore, the inter-chain loop region of BoNT/E is substituted for thenatural loop region occurring in another BoNT having greater therapeuticactivity or duration of action, for example BoNT/A or /B. In anotherembodiment of the invention the loop region of BoNT/E is modified tocontain a proteolytic cleavage site highly specific to a selectedprotease prior to the subcloning. The otherwise highly conserved BoNT/Eloop region would be resistant to indigenous proteases, or thoseencountered within a human, but would retain the ability to be activatedby digestion with the selected protease.

Essentially, the selected protease therefore acts as an “activator” ofneurotoxin toxicity (or modified toxin activity) that can be added atwill following recombinant expression.

The selected protease may be any protease that recognizes a specificamino acid sequence and cleaves a peptide bond near or at that location,but the selected protease is very preferably not a human protease suchas human trypsin, chymotrypsin or pepsin, or a host cell protease.Moreover, the selected protease does not recognize the same amino acidsequence as the indigenous proteases. Finally, the selected proteaseshould not be one expressed by the host cell that contains the plasmidencoding the recombinant neurotoxin.

Any non-human protease recognizing a relatively rare amino acid sequencemay be used, provided that the amino acid recognition sequence is alsoknown. Examples of proteases to be selected as activators may includeany of the following, without limitation: bovine enterokinase, plantproteases such as papain (from Carica papaya) and legumain, insectpapain homolog (from the silkworm Sitophilus zeamatus), and crustacianpapain homolog (decapod), Tobacco etch virus (TEV) protease, whichcleaves the sequences EXXYXQS (SEQ ID NO: 22) and EXXYXQG (SEQ ID NO:23); GENENASE® from Bacillus amyliquifaciens, which cleaves the sequenceHY or YH; and PRESCISSION® protease from human rhinovirus 3C, whichcleaves the amino acid sequence LEVLFQGP (SEQ ID NO: 16). As used above,the letter X indicates any amino acid.

Unless indicated otherwise, the following terms have the followingmeanings in this specification:

By “binding element” is meant an amino acid sequence region able topreferentially bind to a cell surface marker characteristic of thetarget cell under physiological conditions. The cell surface marker maycomprise a polypeptide, a polysaccharide, a lipid, a glycoprotein, alipoprotein, or may have structural characteristics of more than one ofthese. By “preferentially interact” is meant that the disassociationconstant (K_(d)) of the binding element for the cell surface marker isat least one order of magnitude less than that of the binding elementfor any other cell surface marker. Preferably, the disassociationconstant is at least 2 orders of magnitude less, even more preferablythe disassociation constant is at least 3 orders of magnitude less thanthat of the binding element for any other cell surface marker to whichthe neurotoxin or modified neurotoxin is exposed.

The “translocation element” comprises a portion of a clostridialneurotoxin H chain having a translocation activity. By “translocation”is meant the ability to facilitate the transport of a polypeptidethrough a vesicular membrane, thereby exposing some or all of thepolypeptide to the cytoplasm.

In the various botulinum neurotoxins translocation is thought to involvean allosteric conformational change of the heavy chain caused by adecrease in pH within the endosome.

This conformational change appears to involve and be mediated by the Nterminal half of the heavy chain and to result in the formation of poresin the vesicular membrane; this change permits the movement of theproteolytic light chain from within the endosomal vesicle into thecytoplasm. See e.g., Lacy, et al., Nature Struct. Biol. 5:898-902(October 1998).

The amino acid sequence of the translocation-mediating portion of thebotulinum neurotoxin heavy chain is known to those of skill in the art;additionally, those amino acid residues within this portion that areknown to be essential for conferring the translocation activity are alsoknown.

It would therefore be well within the ability of one of ordinary skillin the art, for example, to employ the naturally occurring N-terminalpeptide half of the heavy chain of any of the various Clostridiumtetanus or Clostridium botulinum neurotoxin subtypes as a translocationelement, or to design an analogous translocation element by aligning theprimary sequences of the N-terminal halves of the various heavy chainsand selecting a consensus primary translocation sequence based onconserved amino acid, polarity, steric and hydrophobicitycharacteristics between the sequences.

The “therapeutic element” of the present invention may comprise, withoutlimitation: active or inactive (i.e., modified) hormone receptors (suchas androgen, estrogen, retinoid, perioxysome proliferator and ecdysonereceptors etc.), and hormone-agonists and antagonists, nucleic acidscapable being of being used as replication, transcription, ortranslational templates (e.g., for expression of a protein drug havingthe desired biological activity or for synthesis of a nucleic acid drugas an antisense agent), enzymes, toxins (including apoptosis-inducing or-preventing agents), and the like.

In a preferred embodiment, the therapeutic element is a polypeptidecomprising a clostridial neurotoxin light chain or a portion thereofretaining the SNARE-protein sequence-specific endopeptidase activity ofa clostridial neurotoxin light chain. The amino acid sequences of thelight chain of botulinum neurotoxin (BoNT) subtypes A-G have beendetermined, as has the amino acid sequence of the light chain of thetetanus neurotoxin (TeTx). Each chain contains the Zn⁺⁺-binding motifHis-Glu-x-x-His SEQ ID NO: 17 (N terminal direction at the left)characteristic of Zn⁺⁺-dependent endopeptidases (HELIH SEQ ID NO: 25 inTeTx, BoNT/A /B and /E; HELNH SEQ ID NO: 26 in BoNT/C; and HELTH SEQ IDNO: 27 in BoNT/D).

Recent studies of the BoNT/A light chain have revealed certain featuresimportant for the activity and specificity of the toxin towards itstarget substrate, SNAP-25. Thus, studies by Zhou et al. Biochemistry34:15175-15181 (1995) have indicated that when the light chain aminoacid residue His₂₂₇ is substituted with tyrosine, the resultingpolypeptide is unable to cleave SNAP-25; Kurazono et al., J. Biol. Chem.14721-14729 (1992) performed studies in the presynaptic cholinergicneurons of the buccal ganglia of Aplysia californica using recombinantBoNT/A light chain that indicated that the removal of 8 N-terminal or 32C-terminal residues did not abolish toxicity, but that removal of 10N-terminal or 57 C-terminal residues abolished toxicity in this system.Most recently, the crystal structure of the entire BoNT/A holotoxin hasbeen solved; the active site is indicated as involving the participationof His₂₂₂, Glu₂₂₃, His₂₂₆, Glu₂₆₁ and Tyr₃₆₅. Lacy et al., supra. (Theseresidues correspond to His₂₂₃, Glu₂₂₄, His₂₂₇, Glu₂₆₂ and Tyr₃₆₆ of theBoNT/A L chain of Kurazono et al., supra.) Interestingly, an alignmentof BoNT/A through E and TeTx light chains reveals that every such chaininvariably has these residues in positions analogous to BoNT/A. Kurazonoet al., supra.

The catalytic domain of BoNT/A is very specific for the C-terminus ofSNAP-25 and appears to require a minimum of 17 SNAP-25 amino acids forcleavage to occur. The catalytic site resembles a pocket; when the lightchained is linked to the heavy chain via the disulfide bond betweenCys₄₂₉ and Cys₄₅₃, the translocation domain of the heavy chain appearsto block access to the catalytic pocket until the light chain gainsentry to the cytosol. When the disulfide bond is then reduced, thecatalytic pocket is “opened” and the light chain is fully active.

As described above, VAMP and syntaxin are cleaved by BoNT/B, D, F, G andTeNT, and BoNT/C₁, respectively, while SNAP-25 is cleaved by BoNT/A Eand C1.

The substrate specificities of the various clostridial neurotoxin lightchains other than BoNT/A are known. Therefore, the person of ordinaryskill in the art could easily determine the toxin residues essential inthese subtypes for cleavage and substrate recognition (for example, bysite-directed mutagenesis or deletion of various regions of the toxinmolecule followed by testing of proteolytic activity and substratespecificity), and could therefore easily design variants of the nativeneurotoxin light chain that retain or lack the same or similar activity.

In a particularly preferred embodiment, the single chain neurotoxin orneurotoxin derivative of the invention, altered as indicated above, isfurther modified to remove other incidental endogenous proteolytic sitessuch as those cleaved by trypsin, Arg C protease, chymotrypsin, or hostcell proteases. As indicated below, modification of the primary aminoacid sequences in these regions to confer protease resistance canincrease the yield of the neurotoxin and reduce the toxicity of thesingle chain neurotoxin prior to cleavage and activation.

The amino acid sequences recognized by many proteases, and theircleavage specificity are well-known to those of skill in the art. Thus,both the design of a specific proteolytic cleavage site in the loopregion between the L and H chain portions of the single-chain toxin andthe modification of incidental protease sites in the polypeptide to beprotease-resistant is a routine matter of comparing the specificity andrecognition sequences for various proteins. In the first case, thespecificity of a candidate proteolytic site need not be totallyexclusive, but merely needs to exclude cleavage sites for human and/orhost cell proteases that might be present during the handling, storageand purification of the single chain neurotoxin. Of course, it ispreferable that the protease site is as specific as possible. In thelatter case, the modification of the proteolytic cleavage site need onlybe sufficient to render the site resistant to the activator protease andto human and host cell proteases.

In another preferred embodiment, the recombinant modified single chainneurotoxin is further modified by joining the chain to a binding tagcomprising one member of a specific binding complex. By “specificbinding complex” is meant two or more chemical or biochemical entitiesthat will bind each other under defined environmental conditions andwhich will not significantly bind other chemical or biochemical entitiespresent in the environment under the same conditions. Examples ofmembers of a specific binding complex include, without limitation, anantibody and its antigen, a lectin and its target carbohydrate, anucleic acid strand and its complementary nucleic acid strand, a cellsurface receptor and its ligand, a metal and a compound able to form acoordination or chelation complex with that metal, and the like.

In this embodiment, the binding tag may be joined to the single chaintoxin through a linker, preferably a cleavable linker. Examples ofpossible linkers, while not an exhaustive list, include 1) aliphaticdicarboxylic acids of the formula HOOC—(CH₂)_(n)—COOH, where n=1-12 (maybe linked at a free amino group); 2) HO—(CH₂)_(n)—COOH, where n>10(suitable for attachment at the amino terminus of the polypeptide), 3)substituted polybenzene structures, and 4) a N-hydroxysuccinimide (NHS)ester linker. The use of an linker containing an ester permits cleavageof the ester linker following use in the purification of the singlechain neurotoxin under relatively mild acidic conditions.

Alternatively, and most preferably, the binding tag may comprise some orall of the amino acid sequence of an appropriately chosen polypeptidecoexpressed with the single chain toxin as a fusion protein; suchpolypeptides may comprise, without limitation, the maltose bindingdomain of maltose binding protein (MBP); a His₆ tag (a run of 6histidine residues); the calmodulin binding domain of calmodulin bindingprotein; and the glutathione binding domain ofglutathione-S-transferase. Other polypeptide binding tags are well knownto those of skill in the art, and can easily be adapted for use in thepresent invention.

Additionally, the binding tag may be constructed to have a proteasecleavage site between itself and either the amino terminus or thecarboxyl terminus of the single chain toxin so as be removable followingpurification of the peptide. The proteolytic cleavage site may bedesigned to be cleaved by the same activator protease chosen to nick thesingle chain toxin between the H and L chains.

It is therefore an object of the invention to provide a recombinantactivatable single chain neurotoxin molecule that has reduced toxicitycompared to the native neurotoxin until activated by reaction with anon-clostridial protease. The single chain neurotoxin is more easilypurified, is less dangerous to handle in the purification process, andcan be optionally modified to give the toxin more desirable properties.

It is also an object of the invention to provide an method of making arecombinant activatable single chain neurotoxin by modifying thenucleotide sequence encoding the neurotoxin to replace the native aminoacid proteolytic cleavage sequence separating the H and L chain with anamino acid sequence stable to indigenous clostridial or host cellproteases but susceptible to cleavage by chosen protease in vitro.

It is further an object of the present invention to provide more stableneurotoxin polypeptides through modification of the nucleotide sequenceof the coding region of the H and L chains thereof, removing incidentalproteolytic cleavage sites by causing the replacement of labile aminoacids with other amino acid residues which confer upon the toxinresistance to undesired proteolytic degradation.

Additionally, it is an object of the invention to provide methods ofpurifying recombinant neurotoxins as a single chain by joining theexpressed single chain neurotoxin to a binding moiety comprising partnerof a specific binding complex which can be used in the affinitypurification with the binding partner comprising the other half of thebinding complex. Purification can be performed batch-wise or in achromatography column. The binding moiety may then be removed followingthe affinity step, and separated from the neurotoxin.

It is also an object of the invention to provide single-chainrecombinant modified neurotoxin molecules for use as therapeutic agents.The modified neurotoxin molecules may have an altered target specificityor an altered activity compared to the native neurotoxin from which itis derived, or both.

It is also an object of the invention to provide a single chainactivatable recombinant neurotoxin that may be more easily purified thanthe wild type neurotoxin. Such a neurotoxin permits the large scalepreparation of properly folded highly pure toxin for clinical use.

The following Examples serve to illustrate particular embodiments of theinvention, and do not limit the scope of the invention defined in theclaims in any way.

EXAMPLE 1 Construction of an Expression Vector Containing a Single ChainTeTx Coding Region

The present invention can be exemplified describing the construction ofa plasmid that will express TeTx in E. coli as a single protein that isreadily purified, i.e., by affinity chromatography. TeTx can be chosenas a pilot system because (i) the availability of an excellent vaccinegreatly reduces the risk of its handling and (ii) it is the mostcomprehensively studied of the toxins in terms of expressing HC and LCdomains. However, those of skill in the art will understand that thesame or similar strategies may be employed using any dichain or binarytoxin or other bioactive molecule expressed as a single polypeptide andactivated by proteolytic cleavage. Single chain molecules wereconstructed containing the wild type TeTx L chain and a mutated versionof the TeTx light chain wherein a glutamic acid residue at position 234is changed to an alanine (termed “E234A”, Ala²³⁴, or “the E234A mutantlight chain”), respectively. This latter mutation results in an inactiveTeTx light chain, and a plasmid encoding the E234A mutant light chain(pMAL-E234A) was constructed as described in Li et al., Biochemistry33:7014-7020 (1994)(hereby incorporated by reference herein). Thefollowing protocol is used for the construction of each single-chaintoxin.

The vector pTrcHisA, purchased from Invitrogen, is modified using aStratagene QuickChange® site-directed mutagenesis kit (for site-directedmutagenesis techniques, see e.g., Smith et al., J. Biol. Chem.253:6651-6560 (1979); incorporated by reference herein in its entirety)to create two extra restriction sites (SalI and HindIII) upstream of thenucleotides encoding a pre-existing enterokinase (EK) cleavage site. Theplasmid also contains a translational start codon (ATG) followed by arun of codons encoding 6 histidine residues immediately upstream of theenterokinase cleavage site. A multiple cloning site containing Bam HI,XhoI, Bgl II, Pst I, Kpn I, Eco RI BstB I and Hind III cleavage sites islocated immediately downstream of the EK site; the Hind III site isremoved by site-directed mutagenesis. The following primers are employedto insert the restriction sites (underlined) upstream of the EK cleavagesite:

SEQ ID NO: 1 GACTGGTGGACAGCAAGTCGACCGGAAGCTTTACGACGATGACG,               Sal I  Hind III and SEQ ID NO: 2CGTCATCGTCGTAAAGCTTCCGGTCGACTTGCTGTCCACCAGTC           Hind III  Sal I

The resulting plasmid contains both Sal I and Hind III sites located atthe 5′ side of the nucleotide sequence encoding the bovine enterokinase(EK) cleavage site.

The nucleotide sequence encoding the wild-type TeTx L chain is obtainedfrom plasmid pMAL-LC, described in Li et al., Biochemistry 33, 7014-7020(1994), incorporated by reference herein. The plasmid encodes the TeTxlight chain as a fusion protein with maltose binding protein (MBP)located immediately upstream of the coding sequence for the L chain. TheMBP and L chain portions of the fusion protein are designed to containthe cleavage site for human blood coagulation factor Xa (Ile-Glu-Gly-Argof SEQ ID NO: 28) to facilitate removal of the MBP once affinitypurification has been performed.

The DNA fragment containing the coding sequence of the L chain isexcised from plasmid pMAL-LC by digesting the plasmid with Sal I andHind III, gel purifying the resulting DNA fragment containing the Lchain, and ligating this fragment into plasmid pTrcHisA at the newlycreated Sal I and Hind III sites upstream of the EK site. This fragmentresults in the excission of the maltose binding protein sequences fromthe N terminus of the L chain.

An identical procedure is used to subclone the DNA fragment containing amutant L chain from plasmid pMAL-LC-Ala²³⁴, in which a single amino acidchange is made at amino acid 234 of the L chain, substituting the nativeglutamic acid with alanine. This change is sufficient to abrogate thezinc endopeptidase activity of the L chain, and to render non-toxic areconstituted tetanus toxin containing native H chain and the Ala²³⁴ Lchain.

The DNA fragment containing the H chain is obtained from plasmidpMAL-HC; construction of this vector is described in Li et al., J.Biochem. 125:1200-1208 (1999), hereby incorporated by reference herein.Briefly, the gene encoding the H chain is constructed by assemblingthree DNA fragments containing different portions of the H chain codingsequence which had been cloned into separate plasmids. The fragmentscomprising the amino terminal half of the H are first amplified usingstandard polymerase chain reaction methods (see, e.g., Mullis, U.S. Pat.No. 4,683,202 and Mullis et al., U.S. Pat. No. 4,800,159, bothincorporated by reference herein in their entirety) and the followingprimers: PCR primers a (containing a Xba I cleavage site) and b(containing a Bgl II cleavage site) (SEQ ID NO: 3 and 4, respectively)are used to amplify the H chain fragment contained in a plasmid termedpTet8; PCR primers c (containing a Bgl II cleavage site) and d(containing both a Hind III and a Sal I cleavage site) (SEQ ID NO: 5 and6, respectively) are used to amplify the H chain fragment contained in aplasmid termed pTet14. The nucleotide sequences of these primers areprovided below, with restriction sites underlined.

SEQ ID NO: 3 AATAGATCTAGATCATTAACAGATTTAGGA (a) SEQ ID NO: 4TTCTAAAGATCTATACATTTGATAACT (b) SEQ ID NO: 5 ATGTATAGATCTTTAGAATATCAAGTA(c) SEQ ID NO: 6 ATCGATAAGCTTTTATCAGTCGACCCAACAATCCAGATTTTTAGA (d)

Following PCR amplification and gel purification of the amplified Hchain fragments, each fragment is digested with Bgl II and ligated toyield the complete N terminal half of the H chain coding region. Thisligation product is then digested with Xba I and Hind III and subclonedinto the multiple cloning site of pMAL-c2-T (the plasmid being also cutwith Xba I and Hind III), which is located downstream of the codingregion for MBP and the factor Xa site. pMAL-c2 is a commerciallyavailable vector well known to those of skill in the art. The resultingplasmid is PMAL-H_(N).

The entire H chain coding region is assembled as follows. The pMAL-H_(N)plasmid is digested with Sac I and Sal I to yield the DNA fragmentencoding the N-terminus of the H chain. Plasmid pTet215 is digested withSal I and Bam HI to yield the DNA fragment encoding the H chain carboxylterminus. The vector pMAL-c2-T is digested with Sac I and Bam HI, andligated to the digested H chain fragments, which will assemble in theproper orientation due to the use of distinct endonucleases. Theresulting plasmid is pMAL-HC.

The DNA fragments encoding the H and L chains (including Ala²³⁴ L chain)are cut and purified directly from pMAL-LC or pMALE234A and pMAL-HCconstructs and subcloned into the modified pTrcHisA vector describedabove. The H chain was first ligated into the modified vector at the BamHI site immediately downstream of the EK site, and the resulting plasmidwas gel purified. Following digestion of this plasmid with Hind III andSal I, the L chain was ligated at a position just upstream of the EKcleavage site.

The resulting plasmid construct contains the nucleotide sequenceencoding the single-chain toxin protein, comprising (from amino tocarboxyl terminus): six histidine residues (the His tag), followed bythe L chain, an enterokinase cleavage site, and the H chain. Thetranslated junction between the L and H chains containing the EKcleavage site (DDDDK) is shown below (in the direction from N-terminusto C-terminus) and in FIG. 1.

SEQ ID NO: 7                        EK siteSKLIGLCKKIIPPTNIRENLYNRTA-GEKLYDDDDKDRWGSSR- SLTDLGGELCIKNEDLTFIAEKN     L chain         interchain loop     H chain

To allow expression of the two chains as a single unit, a nucleotidesequence comprising a stop codon present at the 3′ end of the L chaincoding sequence in the pMAL-LC is removed by site-directed mutagenesisusing two primers (SEQ ID NO: 8 and 9), resulting in a single readingframe containing both H and L chains.

SEQ ID NO: 8 AATAGAACTGCAGGAGAAAAGCTTTACGACGATGAC,           TGATAA(deleted stop codon; coding           strand) and SEQ ID. NO: 9GTCATCGTCGTAAAGCTTTTCTCCTGCAGTTCTATT       TTATCA  (deleted stop codon;non-coding strand)

The resulting pTrcHisA-based construct is transformed into E. colistrain JM109 by heat shock using the method of Hanahan, and transformantcolonies are isolated on Luria agar plates containing 100 μg/mlampicillin. Plasmids are purified from these transformants and theinsertions are confirmed by analytical restriction endonucleasedigestion and agarose gel electrophoresis.

EXAMPLE 2 Expression and Physical Characterization of Single-Chain TeTx

Expression of the pTrcHisA-based single chain TeTx construct (undercontrol of a hybrid trp/lac promoter) is induced by addition of 1 mMIPTG (isopropyl thio-galactopyranoside) to a confluent culture of arepresentative transformant clone in 200 ml Luria broth containing 100μg/ml ampicillin and incubating further at 37° C. for 16 hours beforecell harvest by centrifugation.

The cell pellets are resuspended in 30 ml Buffer A (20 mM Na₂PO₄, 500 mMNaCl (pH 7.8)), then lysed by ultrasonication at 4° C., using 10-secondbursts at a medium setting. Insoluble debris is removed bycentrifugation at 9,000×g for 30 min at 4° C., and the supernatantrecovered by centrifugation.

The supernatant containing each single chain construct is incubated for20 minutes at 22° C. with 2 ml of nickel-ion resin (Invitrogen Corp.)for affinity purification by means of chelation between the histidineresidues at the amino terminus of the single chain toxin molecule andthe nickel. The resins were then load onto mini columns and washed with200 ml of washing buffer (20 mM Na₂PO₄, 500 mM NaCl (pH 6.0)) to removeany non-specifically bound material, the recombinant single-chainproteins are eluted on 0.5 ml fractions with 8-15 ml of 100 mM imidazolein Buffer A. The concentration of the eluted single-chains was measuredby Bradford's protein assay (Bio-Rad Laboratories); approximately 1milligram of the fusion protein was recovered.

EXAMPLE 3 SDS-PAGE and Western Blot Analysis of Recombinant Single-ChainTeTx

The single-chain TeTx constructs are grown in Luria broth containingampicillin at 37° C., and aliquots taken both before and after inductionof protein expression with IPTG. Crude cell extracts are prepared forSDS-PAGE by dilution in sample buffer under reducing conditions in thepresence of β-mercaptoethanol (BME). Following SDS-PAGE electrophoresis,the separated proteins are Western-blotted as follows: the proteins areelectrophoretically transferred to a polyvinylidenedifluoride (PVDF)membrane using standard methods (see, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual (2d ed. Cold Spring Harbor Laboratory Press1989), hereby incorporated by reference in its entirety), the membranetreated to reduce background Ig binding, and then probed using ananti-His₆ antibody, followed by detection using an alkalinephosphatase-conjugated secondary antibody and development with a5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate.

As shown in lanes 1 and 2 of FIG. 2A, the Western blot revealed nodetectable TeTx expression before induction of protein synthesis; bycontrast, a single band of approximate molecular weight 150 kDa wasrevealed in the aliquots taken following protein induction (See lanes 3and 4.) In FIG. 2A, lanes 1 and 3 are the WT light chain construct andlanes 2 and 4 contain the E234A mutant construct.

FIG. 2B is a Western blot of IPTG-induced cell extracts from cellstransformed with the E234A construct. Significantly, no discernablelower molecular weight proteolytic cleavage products of the light chainwere observed, providing evidence for the relative stability of thesingle-chain toxin following expression and purification.

FIG. 3 shows the results of a second experiment, in which affinitypurified recombinant single-chain (SC) TeTx is nicked with enterokinaseas follows. Thirty micrograms of purified single chain toxin areincubated with 1 unit of enterokinase in a solution containing 50 mMTris-HCl (pH 8.0), 1 mM CaCl₂ and 0.1% TWEEN-20® (v/v) (polyoxyethylene(20) sorbitan monolaureate). As a control, the recombinant protein isincubated in the same reaction mixture containing no EK. These samples,plus an aliquot of native (non-recombinant) TeTx are subjected toSDS-PAGE in an 8% polyacrylamide gel under either reducing (+BME) ornon-reducing (−BME) conditions. The resulting gel is used both for aWestern blot and subsequent detection using anti-H claim antibodies(FIG. 3B), and direct staining with Coomassie Blue (FIG. 3A).

As indicated by FIG. 3, under non-reducing conditions all three samples(Native TeTx (Lane 1), unnicked recombinant toxin (Lane 2), andenterokinase nicked recombinant toxin (Lane 3)) will migrate as doublets(apparently different conformers that resolve into a single band uponreduction) with essentially indistinguishable apparent molecular weightsof about 150 kDa. The non-reducing gel confirms that 1) high levels ofexpression are obtained, 2) the disulfide bonds linking the light andheavy chains are fully formed, and 3) the recombinant single chain toxinis not subject to observable proteolytic degradation.

By contrast, under reducing conditions wild-type and nicked recombinanttoxin yield an H chain having a molecular weight of about 100 kDa byboth Western blot and Coomassie staining. Additionally, in the Coomassiestained gel, both of these samples also show a lower molecular weightspecies of about 50 kDa, corresponding to the L chain. The wild-type Lchain will migrate with a lower apparent molecular weight than that ofthe recombinant L chain, which has 22 additional amino acid residues dueto the presence of the His₆ moiety and a modified EK cleavagesite-containing interchain junction region. The unnicked recombinanttoxin (Lane 2) migrates as a single band with an apparent molecularweight of about 150 kDa. Notably, no trace of the unnicked toxin is seenin lane 3, indicating the effectiveness of enterokinase treatment.

EXAMPLE 4 In Vitro Toxin-Induced Paralysis by Recombinant Single-ChainTeTx

The biological activity of the recombinant TeTx is also examined andcompared to wild-type toxin using mouse phrenic nerve hemi-diaphragm,since the native toxin is known to cause neuromuscular paralysis, albeitat higher concentrations than act in the CNS. For this experiment, mouseleft phrenic nerve-hemidiaphragm is dissected from mice (T/O strain,4-week old and ˜20 g in weight) and immediately transferred into aclosed circulatory superfusion system containing 10 ml of Krebs-Ringersolution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 23.8 mMNaHCO₄,1.2 mM KH2PO₄, 11.7 mM glucose (pH 7.4)), bubbled with 95% O₂ and5% CO₂ and supplemented with 0.1% (w/v) bovine serum albumin to diminishnon-specific adsorption of the toxins (Li et al., Biochemistry33:7014-7020 (1994)). The hemidiaphragms are kept in a bath containing10 ml Krebs-Ringer buffer at 37° C. for 10 minutes before being exposedto 4 or 10 nM native TeTx (▾ and ∇, respectively) or 10 nM nickedrecombinant TeTx (●) or 10 nM un-nicked recombinant TeTx (

), respectively. (See FIG. 4).

Muscle twitch is evoked by supra-maximal stimulation of the phrenicnerve with bipolar electrodes and recorded via a force-displacementtransducer (Lectromed, UK) connected to an amplifier and computer system(MacLab, AD Instruments, UK). Parameters of nerve stimulation are 0.2 Hzsquare waves of 0.1 msec duration with 1.5-2.5 V amplitude.Toxin-induced paralysis of neuromuscular transmission is quantified asthe time required for nerve-evoked muscle contraction to decrease to 10%(90% reduction) of the original value.

As shown in FIG. 4, 10 nM recombinant nicked TeTx was found to be aspotent as 10 nM native toxin in blocking nerve-induced muscle twitch,with the preparations yielding a 90% reduction in muscle tension inapproximately 170 minutes. Thus, this novel preparation of TeTxexpressed in E. coli at high level as a single-chain, activatablepolypeptide and purified by a simple affinity chromatography step provedto be fully active by all the criteria examined.

By contrast, 10 nM of the unnicked TeTx preparation requireapproximately twice as long to reduce muscle tension, and wasapproximately as active as 4 nM of the wild-type TeTx. As a control,hemidiaphragms incubated with KR buffer and the trace amount ofenterokinase present in the experimental samples were found to shownegligible decrease in muscle tension over 5 hrs.

Thus, this experiment indicates that the unnicked TeTx is considerablyless toxic that either the wild type or recombinant nicked protein invitro.

EXAMPLE 5 Further Modification of Single Chain TeTx to RemoveProteolytic Cleavage Sites Reduces Toxicity of Unnicked RecombinantToxin

While the unnicked recombinant single-chain form of TeTx displaysreduced toxicity as compared to the nicked form, the residual toxinactivity probably arises from activation of the toxin by additionalproteases in vivo. To test this possibility, sites in the single chaintoxin molecule susceptible to proteolytic cleavage by trypsin and Arg Cprotease are identified by incubation of single-chain TeTx with theseenzymes as follows. Fifty micrograms μg of recombinant single chain TeTxis incubated with 4 μg of Arg-C at 37° C. for 4 h; 0.1 μg of trypsin at37° C. for 0.5 h; or buffer without protease as a control. Thesereactions are terminated by the addition of SDS-PAGE sample buffercontaining 0.1% SDS followed by boiling for 5 minutes; then the samplesare subjected to SDS-PAGE, followed by a Western electrophoretictransfer to a polyvinylidenedifluoride (PVDF) membrane. The membrane isblotted with IgG specific for the His₆-tag and detected using ahorseradish peroxidase staining system.

As shown in FIG. 5, the Western blot reveals that trypsin and Arg Cprotease yielded a L chain (and thus a H chain) fragment of the samesize. Additionally, the transfer of a duplicate gel was stained forprotein with Ponceau red and the H chain band of approximate molecularweight 100 kDa was excised from each lane and analysed by N-terminalsequencing.

In the recombinant single-chain TeTx, the LC and HC are linked by 17amino acids (GEKLYDDDDKDRWGSSR SEQ ID NO: 29), followed by the beginningof the H chain sequence (SEQ ID NO: 30). N-terminal amino acidsequencing of the larger fragment produced by both trypsin and Arg Cprotease reveal that first 5 amino acids of the 100 kDa trypsin and ArgC protease cleavage product protein are SLTDL of SEQ ID NO: 20; thus,these proteases appear to cleave the single-chain toxin between the R—Sbond (see FIG. 1) so as to liberate the H chain and the L chaincontaining the EK linker at its C terminus, with this variant thereforeyielding a dichain toxin essentially identical to the EK nicked toxin.

The arginine at the carboxy terminus of the EK linker sequence ismutated by site-directed mutagenesis to a glycine (R496G), and theresulting single chain toxin polypeptide is expressed and purified asabove.

Titration of the 6 micrograms of the R496G mutated single chain (WT LC)toxin and the SC TeTx lacking such a mutation against 0, 0.01, 0.1, 1,10μg/ml of trypsin, followed by SDS-PAGE and staining with CoomassieBrilliant Blue, yields the cleavage pattern seen in FIG. 6. As can beseen, both single chain molecules are susceptible to typsin cleavage;however the R496G mutant yields fewer fragments than the SC toxin notcontaining a mutation in the loop region between the chains. Forexample, while three trypsin peptide bands can clearly be seen near thelight chain band upon trypsin cleavage of the SC WT toxin, only two suchbands are seen in the R496G digests.

The fact that there exist remaining trypsin sites in the R496G mutant SCtoxin probably accounts for the fact that this mutant does not cause thelowering of toxicity as compared to the un-nicked SC toxin; bothpreparations give similar values in the mouse lethality andneuromuscular paralysis assays described above.

A different assay system is used to measure neurotoxin activity towardCNS neurons, the cells naturally affected by TeTx. The cells used arecerebellar neurons; these cells are disassociated from the cerebella of7 day old rats. Neurons are suspended at 1-2×10⁶/mL in medium consistingof 3 parts Basal Eagle Medium and 1 part of a buffer consisting of 40 mMHEPES-NaOH (pH 7.3), 78.4 mM KCl, 37.6 mM D-glucose, 2.8 mM CaCl₂,1.6 mMMgSO₄ and 1.0 mM NaH₂PO₄, as well as 1×N2 supplement, 1.0 mML-glutamine, 60 units/mL penicillin, 60 μg/mL streptomycin and 5% (v/v)dialysed horse serum. One milliliter of this cell suspension is added to22 mm diameter poly-D-lysine coated wells. Cytosineβ-D-arabinofuranoside (Ara-C, 40 μM) is added after 20-24 hours in 5%(v/v) CO₂ culture, and neurons are maintained by weekly replacement ofthe above-noted medium containing 40 μM Ara-C.

For each assay, neurons are cultured for at least 10 days in vitro arewashed four times with O₂-gassed Krebs-Ringer HEPES buffer (KRH, mM: 20HEPES.NaOH pH7.4, 128 NaCl, 5 KCl, 1 NaH₂PO₄,1.4 CaCl₂,1.2 mM MgSO₄, 10D-glucose and 0.05 mg/mL BSA), and 0.5 mL of the latter buffercontaining 0.25 μCi/mL [14C]-glutamine (i.e. the glutamate precursor) isadded. All steps are performed at 37° C. After a 45 minute labelingperiod, the medium is removed and the neurons washed four times asbefore. Control and toxin-treated neurons are incubated for 5 minutes at37° C. in KRH buffer containing either 1.4 mM Ca²⁺ or 0.5 mM EGTA (i.e.to assess Ca²⁺-independent release); aliquots are then removed andretained for assessment of [¹⁴C]-glutamate content by scintillationcounting. Immediately after removal of the above basal medium, amodified KRH buffer containing 50 mM KCl (containing a lowered 83 mMNaCl content in order to maintain a normal osmotic potential) and either1.4 Ca²⁺ or 0.5 mM EGTA are added for a 5 minute stimulation period.Finally, neurons were solubilized with 20 mM EGTA.NaOH pH 7.5 containing1% (w/v) SDS, and aliquots subjected to scintillation counting in orderto calculate their remaining radioactive contents. The amounts of¹⁴C-glutamate in basal and stimulated samples are expressed aspercentages relative to the calculated total cell content. Thepercentage [¹⁴C]-glutamate contents in EGTA-containing buffer aresubtracted from the values recorded in Ca²⁺-containing samples in orderto calculate the relevant Ca²⁺-dependent component of release and inturn the latter basal readings are subtracted from values obtained for50 mM KCl samples to yield the K⁺-evoked Ca²⁺-dependent glutamaterelease component.

FIG. 8 demonstrates the ability of the recombinant toxin to inhibitneurotransmitter release. Cerebellar neurons, maintained for 10 days invitro, were washed twice with ice-cold KRH buffer containing 5 mM Mg²⁺and 0.5 mM Ca²⁺, then exposed in this buffer to the specifiedconcentrations of (●) native TeTx, (∘) EK-nicked TeTx R496G, (▾) singlechain unnicked TeTx, or (∇) EK-nicked TeTx E234A for 60 min at 4° C.(see FIG. 8). Native TeTx (0.2 nM) was then added to the wells specifiedand, after an additional 30 min, the neurons were washed three timeswith ice-cold KRH buffer and incubated for 30 min at 37° C. Subsequentassessment of K⁺-evoked Ca²⁺-dependent neurotransmitter release wasperformed as detailed above. The results of this assay are shown in FIG.8.

When cerebellar neurons are exposed to nicked recombinant TeTx, adose-dependent inhibition of Ca⁺⁺ dependent transmitter release is seenwith a potency similar to the native toxin. Nicked recombinant SC TeTx,both WT and R496G, gave similar values in this assay. Thus, while toxinactivity in the unnicked single chain molecule is not abrogated throughthe removal of a single trypsin cleavage site, the removal of additionalsuch sites is feasible in regions of the single chain toxin to achievean activatable single-chain proform of the toxin that exhibits evenlower toxicity unless activated in vitro, when its full activity can beachieved.

EXAMPLE 6 Protease-Deficient TeTx Mutant Antagonises the Actions of TeTxon Peripheral and Central Neurons

Table 1 shows the tabulated results of the indicated TeTx constructstested in three assays of toxin activity: ability to cleave the HV62peptide (which measures proteolytic activity only); neuromuscularparalysis (which is an indication of the toxin molecules' ability toenter the cell and thence to inhibit neurotransmitter release), andmouse lethality upon intraperitoneal injection of the various toxinconstructs. The first two of these assays was performed as describedabove.

The mouse lethality assay was performed essentially as follows: Samplesof recombinant purified single-chain TeTx, R496G mutant TeTx, and E234Amutant TeTx are each divided into two aliquots and one aliquot treatedwith enterokinase to nick the toxin. All samples are serially dilutedinto 50 mM phosphate buffer (pH 7.0), 150 mM NaCl and 0.25% (w/v) bovineserum albumin (BSA), and the toxin preparations are injected into miceintraperitoneally.

As shown in Table 1, the native and nicked TeTx preparations werecomparably active in the mouse lethality assay, having an LD₅₀ of about1×10⁸/mg. The unnicked recombinant toxin and unnicked R496G mutant wereboth about half as active. Finally, the nickedf E234A proteolyticallyinactive toxin was less than 5×10⁷ fold less active.

TABLE 1 Biological Activity of SC TeTx wild type and mutants (E234A andR496G) before and after nicking with enterokinase Time (min.) Initialrate of for 10 nM cleavage^(a) of HV62 Mouse to cause 90% Purified TeTx(nmol · min⁻¹mg⁻¹) lethality^(b) neuromuscular preparations [Relativerate (%)] (LD50/mg) paralysis Native 20.3 ± 0.91 1 × 10⁸ 145 Un-nickedSC  8.0 ± 0.03 0.5 × 10⁸   260 WT Nicked^(c) SC WT 22.7 ± 3.37 1 × 10⁸150 Un-nicked SC 11.7 ± 0.6  0.5 × 10⁸   250 ± 15 R496G Nicked^(c) SC52.3 ± 4.9  1 × 10⁸ 135 ± 10 R496G Un-nicked SC ≦0.01^(d) Not tested Nottested E234A Nicked^(c) SC ≦0.01^(d) <50 No detectable E234A activity^(a)Initial rates of proteolysis were measured using the RP-HPLC-basedmethod detailed in Foran et al. (1994). Incubations with 15 μM of asynthetic peptide corresponding to residues 33 to 94 of human VAMP-2(HV62) were performed at 37° C. in 50 mM HEPES, NaOH pH 7.5 containing 2mM DTT 0.2 mg · ml⁻¹ BSA and 50 μM ZnCl₂, using the appropriateconcentration of each reduced toxin preparation required to proteolyze10-15% of the substrate during a 30 min period. Data are means (±S.D.; n= 4). ^(b)LD₅₀ is the amount of toxin that killed 50% of the injectedmice within 4 days. ^(c)Toxin preparations were nicked with EK (1unit/30 μg) at 22° C. for 1 h. ^(d)This v^(o) value represents thedetection limits of the RP-HPLC assay; no proteolysis of HV62 wasobserved using prolonged incubations.

Purified SC E234A TeTx, in which the catalytic E at position 234 wasreplaced by an A, failed to show any detectable proteolysis of a peptidecontaining residues 33 to 94 of human VAMP-2 (termed HV62), eitherbefore or after nicking with EK. Accordingly, nicked TeTx E234A provedto be devoid of toxicity in mice and unable to inhibit transmitterrelease at the neuromuscular junction or from cerebellar neurons.

Importantly, however, this mutant toxin retained the ability to bind tothe cell surface receptors on peripheral and central neurons.Pre-incubation of cerebellar neurons with nicked (10-60 nM) or unnicked(7-40 nM) TeTx E234A at 4° C. followed by the addition of 0.2 nM nativetoxin, antagonized the native toxin's inhibition of transmitter releaseat 37° C. to similar extents (FIG. 7).

As demonstrated in FIG. 9, exposure of mouse diaphragm to 100 nM TeTxE234A at 4° C. for 60 minutes prior to adding 1 nM native toxinprolonged the time taken to cause neuromuscular paralysis.

Mouse phrenic-nerve hemi-diaphragm was incubated in KR at 37° C. with 20nM recombinant TeTx E234A (Δ) whilst stimulating the nerve (0.2 Hz,1.5-2.5v) and recording muscle tension. For assessing competition,hemi-diaphragms were incubated for 60 minutes at 4° C. with MKRcontaining 0.1% BSA only (

), or the latter plus 100 nM nicked TeTx E234A (∘), before the additionof 1 nM native TeTx. Following 30 minutes exposure to the latter, thetissues were washed three times with MKR and twice with KR. Thetemperature was raised to 37° C. and the nerve stimulated with recordingof the evoked muscle twitch, as outlined above. This apparentcompetition for toxin binding by the mutant seen with both tissuesdemonstrates that the recombinant dichain TeTx exhibits much higheraffinity for the cell surface receptors than the heavy chain or H_(c) ofTeTx alone. These results suggest that the conformation of therecombinant dichain TeTx has high affinity to the cell surface receptor.

Moreover, and very significantly, these data demonstrate thatrecombinant molecules can be made according to the inventive methods ofthe present patent application having specific binding for the samecellular receptor as TeTx. However, such molecules may, like the E234Amutant, be inactive as toxin molecules but will retain the ability to betaken up by the target cell; thus serving as potential transportermolecules.

EXAMPLE 7 Expression of Single Chain BoNT/A

Using methods similar to those described above, DNA fragments containingthe BoNT subtype A neurotoxin H and L chains were ligated together,separated by the EK cleavage site. This single-chain toxin codingsequence was inserted into a variety of expression vectors containingdifferent N terminal sequences and promoters, as shown in Table 2,below.

TABLE 2 Tag Size Fusion Pro- (amino Size E. coli Vector moter Fusion Tagacids) (kDa) strain pTrcSCPHY trc Poly His 18 150 JM109 pCalSCPHY T7Calmodulin 31 154 BL21 binding protein (DE3) pETSCPHY T7 Poly His 32 154BL21 (DE3) pGEXSCPHY tac Glutathione-S- 224 177 JM109 tranferase pMALPHYtac Maltose 390 193 JM109 Binding Protein

The “fusion tags” each comprised a member of a specific binding complexas a purification aid and to improve the solubility and stability of theexpressed protein. These plasmids were transformed into the E. colistrains indicated in Table 2 and expression of the single-chain toxinwas monitored.

In another experiment, the single-chain BoNT/A construct was insertedinto plasmid pMAL-c2 between the Bam HI and Hind III restriction sites,resulting in a coding sequence for a fusion polypeptide containing themaltose binding protein at the N terminus, followed by a Factor Xacleavage site. Transformant JM 109 colonies were selected in Luria brothcontaining ampicillin. Expression was induced by the addition of IPTG toa final concentration of 0.3 mM. As for the TeTx construct, aliquots ofthe cell culture were collected before and after induction, the cells ineach sample lysed by sonication, and the supernatant prepared forSDS-PAGE under both reducing and non-reducing conditions. Followingelectrophoresis to separate the proteins according to apparent molecularweight, the gel was subjected to a Western blot using an antibody raisedagainst the H chain of BoNT/A. The Western blot resulted in theappearance of an immunologically reactive single-chain toxin band ofapparent molecular weight approximately 200 kDa. Further modificationsof the single-chain BoNT molecule to eliminate fortuitous proteasecleavage sites (similar to those modifications made at the TeTx sitelabile to trypsin and Arg C protease, described above) will result ineven greater stability of the single-chain BoNT/A molecule.

EXAMPLE 8 Construction of a Plasmid Vector Expressing BoNT/E

A plasmid expressing a single-chain recombinant version of theneurotoxin from Clostridium botulinum subtype E (strain Beluga) (BoNT/E)was constructed as follows. PCR primers were designed based on the EMBLdatabase cDNA sequence of the BoNT/E neurotoxin (Genbank accessionnumber X62089) This nucleotide sequence is represented herein as SEQ IDNO: 10.

gaattcaagt agtagataat aaaaataatg ccacagattt ttattattaa taatgatatatttatctcta actgtttaac tttaacttat aacaatgtaa atatatattt gtctataaaaaatcaagatt acaattgggt tatatgtgat cttaatcatg atataccaaa aaagtcatatctatggatat taaaaaatat ataaatttaa aattaggaga tgctgtatat gccaaaaattaatagtttta attataatga tcctgttaat gatagaacaa ttttatatat taaaccaggcggttgtcaag aattttataa atcatttaat attatgaaaa atatttggat aattccagagagaaatgtaa ttggtacaac cccccaagat tttcatccgc ctacttcatt aaaaaatggagatagtagtt attatgaccc taattattta caaagtgatg aagaaaagga tagatttttaaaaatagtca caaaaatatt taatagaata aataataatc tttcaggagg gattttattagaagaactgt caaaagctaa tccatattta gggaatgata atactccaga taatcaattccatattggtg atgcatcagc agttgagatt aaattctcaa atggtagcca agacatactattacctaatg ttattataat gggagcagag cctgatttat ttgaaactaa cagttccaatatttctctaa gaaataatta tatgccaagc aatcaccgtt ttggatcaat agctatagtaacattctcac ctgaatattc ttttagattt aatgataatt gtatgaatga atttattcaagatcctgctc ttacattaat gcatgaatta atacattcat tacatggact atatggggctaaagggatta ctacaaagta tactataaca caaaaacaaa atcccctaat aacaaatataagaggtacaa atattgaaga attcttaact tttggaggta ctgatttaaa cattattactagtgctcagt ccaatgatat ctatactaat cttctagctg attataaaaa aatagcgtctaaacttagca aagtacaagt atctaatcca ctacttaatc cttataaaga tgtttttgaagcaaagtatg gattagataa agatgctagc ggaatttatt cggtaaatat aaacaaatttaatgatattt ttaaaaaatt atacagcttt acggaatttg atttacgaac taaatttcaagttaaatgta ggcaaactta tattggacag tataaatact tcaaactttc aaacttgttaaatgattcta tttataatat atcagaaggc tataatataa ataatttaaa ggtaaattttagaggacaga atgcaaattt aaatcctaga attattacac caattacagg tagaggactagtaaaaaaaa tcattagatt ttgtaaaaat attgtttctg taaaaggcat aaggaaatcaatatgtatcg aaataaataa tggtgagtta ttttttgtgg cttccgagaa tagttataatgatgataata taaatactcc taaagaaatt gacgatacag taacttcaaa taataattatgaaaatgatt tagatcaggt tattttaaat tttaatagtg aatcagcacc tggactttcagatgaaaaat taaatttaac tatccaaaat gatgcttata taccaaaata tgattctaatggaacaagtg atatagaaca acatgatgtt aatgaactta atgtattttt ctatttagatgcacagaaag tgcccgaagg tgaaaataat gtcaatctca cctcttcaat tgatacagcattattagaac aacctaaaat atatacattt ttttcatcag aatttattaa taatgtcaataaacctgtgc aagcagcatt atttgtaagc tggatacaac aagtgttagt agattttactactgaagcta accaaaaaag tactgttgat aaaattgcag atatttctat agttgttccatatataggtc ttgctttaaa tataggaaat gaagcacaaa aaggaaattt taaagatgcacttgaattat taggagcagg tattttatta gaatttgaac ccgagctttt aattcctacaattttagtat tcacgataaa atctttttta ggttcatctg ataataaaaa taaagttattaaagcaataa ataatgcatt gaaagaaaga gatgaaaaat ggaaagaagt atatagttttatagtatcga attggatgac taaaattaat acacaattta ataaaagaaa agaacaaatgtatcaagctt tacaaaatca agtaaatgca attaaaacaa taatagaatc taagtataatagttatactt tagaggaaaa aaatgagctt acaaataaat atgatattaa gcaaatagaaaatgaactta atcaaaaggt ttctatagca atgaataata tagacaggtt cttaactgaaagttctatat cctatttaat gaaaataata aatgaagtaa aaattaataa attaagagaatatgatgaga atgtcaaaac gtatttattg aattatatta tacaacatgg atcaatcttgggagagagtc agcaagaact aaattctatg gtaactgata ccctaaataa tagtattccttttaagcttt cttcttatac agatgataaa attttaattt catattttaa taaattctttaagagaatta aaagtagttc agttttaaat atgagatata aaaatgataa atacgtagatacttcaggat atgattcaaa tataaatatt aatggagatg tatataaata tccaactaataaaaatcaat ttggaatata taatgataaa cttagtgaag ttaatatatc tcaaaatgattacattatat atgataataa atataaaaat tttagtatta gtttttgggt aagaattcctaactatgata ataagatagt aaatgttaat aatgaataca ctataataaa ttgtatgagagataataatt caggatggaa agtatctctt aatcataatg aaataatttg gacattcgaagataatcgag gaattaatca aaaattagca tttaactatg gtaacgcaaa tggtatttctgattatataa ataagtggat ttttgtaact ataactaatg atagattagg agattctaaactttatatta atggaaattt aatagatcaa aaatcaattt taaatttagg taatattcatgttagtgaca atatattatt taaaatagtt aattgtagtt atacaagata tattggtattagatatttta atatttttga taaagaatta gatgaaacag aaattcaaac tttatatagcaatgaaccta atacaaatat tttgaaggat ttttggggaa attatttgct ttatgacaaagaatactatt tattaaatgt gttaaaacca aataacttta ttgataggag aaaagattctactttaagca ttaataatat aagaagcact attcttttag ctaatagatt atatagtggaataaaagtta aaatacaaag agttaataat agtagtacta acgataatct tgttagaaagaatgatcagg tatatattaa ttttgtagcc agcaaaactc acttatttcc attatatgctgatacagcta ccacaaataa agagaaaaca ataaaaatat catcatctgg caatagatttaatcaagtag tagttatgaa ttcagtagga aattgtacaa tgaattttaa aaataataatggaaataata ttgggttgtt aggtttcaag gcagatactg tcgttgctag tacttggtattatacacata tgagagatca tacaaacagc aatggatgtt tttggaactt tatttctgaagaacatggat ggcaagaaaa ataaaaatta gattaaacgg ctaaagtcat aaattccThe forward primer had the following nucleotide base sequence:

SEQ ID NO: 11 CCCGGATCC CCA AAA ATT AAT AGT TTT AAT TAT AAT Gwhere the BamHI endonuclease site is underlined and the sequence of thelight chain minus the start codon is in bold. The inverse primer had thesequence:

SEQ ID NO: 12 CCCCTGCAG tca TTT TTC TTG CCA TCC ATG TTC TTCwhere the PstI endonuclease site is underlined, the end of the codingregion of the heavy chain is in bold, and the stop codon is in lowercase. These primers were made using standard DNA synthesis methodology.

The two primers were used in a PCR reaction containing different amountsof Clostridium botulinum type E (strain beluga) chromosomal DNA. The PCRreaction employed a DNA polymerase with proofreading activity (Pfx DNApolymerase, obtained from Life Technology) in order to avoid sequenceerrors in the amplified gene. The amplification reaction conditions wereas follows: 30 cycles of: a 45 second denaturation at 95° C., followedby a 45 second annealing step at 56° C., followed by a primer extensionreaction for 3 minutes 48 seconds at 68° C.

The PCR product was digested with BamHI and HindIII, and the digestsubjected to agarose gel electrophoresis. Staining of the agarose gelwith ethidium bromide revealed a major DNA fragment of approximately 3.5kilobases (see FIG. 10). The band containing this fragment was excisedfrom the gel, and the DNA purified from the agarose and ligated to BamHIand HindIII-cut pQE30 vector (Qiagen). The resulting ligated plasmid wasused to transform E. coli strain JM 109 as described above, and thetransformants plated onto selective LB agar plates. Several clones wererecovered and the presence of the correct BoNT/E DNA insert checked byrestriction digest. The resultant construct contains the BoNT/E gene(minus the first methionine) fused to the His₆ tag of the pQE30 vector,and contains 2 extra amino acid residues (glycine, serine), which arecontributed by the engineered BamHI site.

EXAMPLE 9 Construction of a Proteolytically-Inactive Mutant of BoNT/E bySite Directed Mutagenesis

By mutating the glutamic acid at position 212 (within the active site)of the BoNT/E polypeptide construct to glutamine, aproteolytically-inactive and non-toxic single chain BoNT/E polypeptidewas obtained.

The glutamine replacement was introduced on the forward primer usingroutine site directed mutagenesis methods. The mutagenic DNA primer hadthe sequence

SEQ ID NO: 13 cag TTA ATA CAT TCA TTA CAT GGA CTA TAT Gwhere the codon encoding glutamine at position 212 is indicated in smallletters.

An inverse PCR reaction was performed using the above primer, along withthe reverse primer

SEQ ID NO: 14 ATG CAT TAA TGT AAG AGC AGG ATC TTAnd Pfx DNA polymerase (Life Technology) as above. The PCR template wasthe wild-type single-chain BoNT/E construct (termed pQEESCwt). Thecycling parameters (30 cycles) were as follows: 1) a 45 seconddenaturation step at 95° C.; 2) a 45 second annealing step at 56° C.;and 3) a 7 minute 10 second extension step at 68° C.

At the end of the amplification reaction, the DNA template was digestedby the restriction enzyme DpnI to permit selection of mutated clonesonly. After subjecting the PCR product to agarose gel electrophoresis, aband of approximately 7 kilobases was removed and the DNA purified andused for self-ligation in the presence of T4 DNA ligase (Promega) andpolynucleotide kinase (Promega) to permit phosphorylation of the PCRproduct. The ligation mixture was used to transform E. coli strainDH10B, and the transformants plated onto selective agar plates. Thepresence of the correct plasmide construct was verified in severalrepresentative transformants by restriction digest and the mutationconfirmed also by DNA sequencing. FIG. 11 shows the protocol forconstruction of the mutant BoNT/E plasmid, and an ethidiumbromide-stained agarose gel of the PCR reaction mixture (lanes 2 and 3)versus molecular weight markers (lane 1).

EXAMPLE 10 Purification of Single Chain Recombinant BoNT/E

The presence of the histidine tag at the N-terminus of the expressedprotein allowed a single-step purification of the recombinant neurotoxinby metal-affinity chromatography.

The E. coli strain M15 (Qiagen) was used for expression of the BoNT/Esingle-chain construct. This strain carries an endogenous plasmid(pREP4, kanamycin resistant) containing a region encoding the lac I^(q)repressor gene in order to prevent transcription of the neurotoxin geneprior to induction with IPTG. The pQE30 vector contains a T5bacteriophage RNA polymerase promoter, which is also recognized by E.coli RNA polymerase.

A colony of M15 cells containing pQEESCwt was grown at 37° C. overnightin 5 ml of 2TY medium containing 0.1 mg/ml ampicillin; 0.025 mg/mlkanamycin and 0.2% glucose (w/v), and the resultant culture used toinoculate 500 ml of the same medium. When this second culture reached anoptical density of 0.5-0.8 at 600 nm, IPTG was added to a finalconcentration of 0.3 mM and the culture incubated at 25° C. overnight topermit expression of the neurotoxin.

Subsequent centrifugation of the culture yielded ˜2.3 g of wet cellpellet which was resuspended in 10 ml of extraction buffer (20 mM HepespH 7.0, 300 mM NaCl, 5 mM benzamidine, 2 μM pepstatin and 2 μM E-64).Lysozyme was added to a final concentration of 0.25 mg/ml, and the cellsuspension incubated on ice for 60 minutes. Approximately 0.5 ml ofglass beads (0.1 mm diameter from Biospec) was added to the cellsuspension, followed by vortexing for 2 minutes to break the cells.Cell-free extracts was obtained by centrifugation at 10,000×g for 30minutes at 4° C. The supernatant was incubated with 0.5 ml of TALON®cobalt metal affinity agarose resin (Clontech) pre-washed withextraction buffer in a rocking platform for 45 minutes at 4° C. Theresin was then loaded into a disposable chromatography column and washedtwice with 10 bed volumes of wash buffer (20 mM Hepes pH 7.0, 300 mMNaCl, 2 mM imidazole) before eluting the bound neurotoxin in 6 bedvolumes of elution buffer (20 mM Hepes pH 7.0, 300 mM NaCl, 150 mMimidazole).

The elute was dialyzed overnight at 4° C. against 10 mM Hepes (pH 7.0)containing 150 mM NaCl and concentrated by centrifugal filtration (MWcutoff 10 KDa) to a final concentration of 1 mg/ml protein.

As shown in FIG. 12, the purity of the affinity-purified toxin wasdemonstrated by SDS-PAGE under reducing conditions, followed byCoomassie staining and Western-blotting, detecting the N-terminus with amouse monoclonal anti-His antibody from Quiagen (diluted 2000 fold).Enhanced Chemiluminescence solutions (Santa Cruz) and mouse secondaryhorseradish peroxidase (affinity purified from Sigma) were used fordetection of bound antibody. Approximately 2 μg of protein samples wereloaded per well.

EXAMPLE 11 Trypsin Activation of Purified Recombinant BoNT/ESingle-Chain Polypeptide

Purified BoNT/E single-chain neurotoxin polypeptide samples wereactivated by nicking the single chain with trypsin (1.5 μg/ml finalconcentration) for 60 minutes at a concentration of 1 mg toxin/ml in 10mm Hepes (pH 7.0), 150 mM NaCl. Following the reaction, the trypsin wasinactivated using 0.5 mM PMSF and 10 μg trypsin inhibitor/ml. Thequality of the trypsinization was assessed and verified by SDS-PAGEunder both reducing and non-reducing conditions, then staining withCoomassie staining and Western blotting the polyacrylamide gel using amouse monoclonal anti-His antibody (Quiagen, diluted 2000-fold) and amouse monoclonal anti-H_(C) IgG (diluted 26-fold). As shown in FIG. 13,the Commassie-stained nicked protein resolves into two bands underreducing conditions, while the heavy and light chains remaindisulfide-linked under non-reducing conditions, similar to the nativetoxin. The antibody-detected recombinant heavy chain is of approximatelyidentical size as its wild-type Clostridium counterpart, whereas therecombinant light chain migrates at a slightly higher molecular weightcompared to the native protein. This latter characteristic is due to theextra residues provided by the His₆ tag at the N-terminus.

EXAMPLE 12 Recombinant BoNT/E is Proteolytically Active

Stock solutions (1 μM) of native nicked BoNT/E toxin, un-nickedsingle-chain recombinant toxin, nicked di-chain recombinant toxin, andnicked mutant (E212Q) BoNT/E were prepared in HEPES-buffered saline(HBS, 150 mM NaCl, 10 mM HEPES, pH 7.4, 10 μg/ml BSA). These sampleswere incubated for 30 minutes at 37° C. in the absence or presence of 20mM DTT, and then serially diluted in 0.02 ml of HBS to the finalconcentrations shown in FIG. 14.

A recombinant peptide containing amino acids 140-205 of SNAP-25 fused toglutathione-S-transferase (termed GST-SNAP-25 [140-205]) was used as aprotease substrate to test the proteolytic activity of the recombinantBoNT/E polypeptides. Ten micrograms this protease substrate wasincubated with the toxin samples. The digestion reaction was allowed toproceed for 30 minutes at 37° C. in the absence or presence of 2 mM DTT,and stopped by addition of SDS-PAGE sample buffer followed by boilingfor 5 minutes.

The resultant samples were analyzed by SDS-PAGE (3 μg of GST-SNAP-25[140-205] per lane) and silver staining. As FIG. 14 demonstrates, evenunnicked recombinant single chain toxin retains proteolytic activity. Asexpected, the mutant E212Q BoNT/E construct has no detectableproteolytic activity. FIG. 14 shows only the GST-SNAP-25[140-205] bands.

EXAMPLE 13 Nicking Makes Recombinant BoNT/E Fully Functional

Cerebellar neurons maintained for 10 days in culture (2×10⁶/22 mmdiameter well) were washed with Krebs-Ringer HEPES (KRH) buffer, thenexposed to the specified concentrations of BoNT/E native (●),trypsin-nicked recombinant (

), or un-nicked single-chain (▾) BoNT/E. (See FIG. 15). After 60 minutesat 37° C., the toxin-containing buffer was removed and the cells werewashed twice, then incubated with KRH buffer containing 0.25 μCi/ml[¹⁴C]-labeled glutamine (i.e. the glutamate precursor). After 45minutes, the latter medium was removed and the neurons were washed fourtimes at 37° C. prior to assessment of transmitter glutamate release.Control and toxin-treated neurons were incubated for 5 minutes at 37° C.in KRH buffer containing either 1.4 mM Ca²⁺ or 0.5 mM EGTA to assessCa²⁺-independent release; aliquots were then removed for determinationof their [¹⁴C]-glutamate content (see below).

Immediately after removal of the basal medium, KRH buffer containing 50mM KCl and either 1.4 mM Ca²⁺ or 0.5 mM EGTA was added; as before,aliquots were removed for [¹⁴C]-glutamate assay after a 5 minutestimulation period. Finally, neurons were solubilized with 20 mMEGTA.NaOH pH 7.5 containing 1% (w/v) SDS and aliquots were removed todetermine the amounts of radioactivity remaining within the cells. Theamount of [¹⁴C]-glutamate in each of the samples was assayed byscintillation counting and the levels released under basal andstimulated conditions were expressed as percentages relative to thecalculated total cell content.

The percent [¹⁴C]-glutamate content in the EGTA-containing buffer foreach sample was subtracted from the values recorded in Ca²⁺-containingKRH samples in order to obtain the Ca²⁺-dependent component of release,and the latter basal readings were subtracted from values obtained for50 mM KCl samples to yield K⁺-evoked Ca²⁺-dependent release. The values,thus, obtained from toxin-treated neurons are expressed relative totoxin-free controls.

FIG. 15 shows that, despite retaining proteolytic activity, theun-nicked recombinant BoNT/E has markedly less activity than either thenative BoNT/E or the nicked recombinant version. This finding mayreflect the inability of the un-nicked toxin to adequately enter thetarget cell. Additionally, the nicked recombinant version appears to bemore effective in inhibiting glutamate release than the native toxin.

EXAMPLE 14 Recombinant BoNT/E has a Neuromuscular Paralytic ActivityEquivalent to that of the Native Toxin at Mouse Neuromuscular Endplates:Nicking Increases Potency

Mouse phrenic-nerve hemi-diaphragms were bathed in KR supplemented with0.1% BSA and saturated with 95% O₂/5% CO₂. The phrenic nerves werestimulated (0.2 Hz, 1.5-2.5 mV) and nerve evoked muscle tension wasrecorded before and after the addition of (FIG. 16A) 0.2 nM recombinantnicked BoNT/E (

) Or 0.2 nM native BoNT/E (□), and (FIG. 16B) 1 nM recombinant un-nicked(

), 1 nM recombinant nicked (●) or 0.05 nM recombinant nicked (∇) BoNT/E.As shown in FIGS. 16A and 16B, the recombinant nicked BoNT/E is aneffective paralytic agent, displaying greater activity in this assaythat the native toxin. The un-nicked toxin displays significantly loweractivity than the nicked toxin in this assay.

The neuromuscular paralytic activity of recombinant nicked BoNT/E wasalso demonstrated in mice by intra-muscular injection into hind-limbmuscles. This resulted in paralysis, as assessed by the toe spreadreflex assay, with a pattern of symptoms typical of botulism.

The in vivo neurotoxicity of the nicked, recombinant neurotoxin wasestablished, by injecting the toxin into mice, to have a specificneurotoxicity of less than 10⁷ mouse LD₅₀ units per mg.

EXAMPLE 15 The BoNT/E E212Q Protease Inactive Mutant AntagonisesBoNT/E-induced Neuroparalysis

A mouse phrenic-nerve hemi-diaphragm was exposed to 10 nM BoNT/E E212Qin KR medium, the nerve was stimulated and evoked muscle tension wasrecorded. As indicated by FIG. 17, the BoNT E212Q mutant does notinhibit neurotransmission, as determined by its failure to reducenerve-evoked muscle tension (

). To assess the ability of this non-toxic mutant to antagonise theactivity of the native toxin, mouse phrenic-nerve hemi-diaphragms werebathed for 60 minutes at 4° C. in MKR supplemented with 0.1% BSA andsaturated with 95% O₂/5% CO₂, without (□) or with (Δ) the inclusion of 5nM BoNT/E E212Q. Native nicked BoNT/E was added to each bath (0.05 nMfinal) and the tissues were incubated for a further 30 min. Thenerve-muscles were then washed three times each with MKR followed by KR,before the temperature was raised to 37° C., the nerve stimulated andevoked muscle tension recorded.

As shown in FIG. 17, the onset of native BoNT/E activity in this assaywas delayed and antagonized when the phrenic-nerve hemi-diaphragms arepreincubated with the E212Q protease inactive mutant, thereby indicatingthat the recombinant mutant faithfully binds to the same cell surfacereceptor as does the native toxin. Thus, the methods of the presentpatent application can be used to produce recombinant and modifiedtoxins having fully functional receptor binding domains, andBoNT-related transported molecules for the intracellular delivery oftherapeutic agents.

Those of skill in the art will understand that the Examples providedherein describe preferred compositions and methods, and that a varietyof different cloning strategies, protease cleavage sites, and specificbinding complex members may be employed in the practice and use of thepresent invention while remaining within the invention's scope.Additionally, different dichain or binary toxin molecules and modifiedversions thereof (for example, BoNT/B-E and modified variants thereof)may be used as the basis for the methods and compositions of the presentinvention.

What is claimed is:
 1. A cleavable single-chain polypeptide comprising:a) a first amino acid sequence region comprising i) a first domaincomprising a botulinum neurotoxin heavy chain binding element able topreferentially interact with a target cell surface marker underphysiological conditions; and ii) a second domain comprising a botulinumneurotoxin heavy chain translocation element able to facilitate thetransfer of said single-chain polypeptide across a vesicular membrane;and b) a second amino acid sequence region comprising a botulinumneurotoxin light chain therapeutic element having SNARE-proteinsequence-specific endopeptidase activity; c) a third amino acid sequenceregion comprising a protease cleavage site, wherein the proteasecleavage site is cleaved by a non-human Enterokinase or a tobacco etchvirus protease; wherein said first and second amino acid sequenceregions are separated by said third amino acid sequence region.
 2. Thepolypeptide of claim 1, wherein said polypeptide further comprises afourth region comprising a target-binding portion of a binding tag. 3.The polypeptide of claim 2, wherein said target-binding portioncomprises a His6, a monoclonal antibody, a maltose binding protein, aglutathione-S-transferase, a protein A, or a calmodulin binding protein.4. The polypeptide of claim 1, wherein said botulinum neurotoxin heavychain binding element is a botulinum neurotoxin serotype A neurotoxinheavy chain, a botulinum neurotoxin serotype B neurotoxin heavy chain, abotulinum neurotoxin serotype C1 neurotoxin heavy chain, a botulinumneurotoxin serotype D neurotoxin heavy chain, a botulinum neurotoxinserotype E neurotoxin heavy chain, a botulinum neurotoxin serotype Fneurotoxin heavy chain and a botulinum neurotoxin serotype G neurotoxinheavy chain.
 5. The polypeptide of claim 1, wherein said botulinumneurotoxin heavy chain translocation element is a botulinum neurotoxinserotype A neurotoxin heavy chain, a botulinum neurotoxin serotype Bneurotoxin heavy chain, a botulinum neurotoxin serotype C1 neurotoxinheavy chain, a botulinum neurotoxin serotype D neurotoxin heavy chain, abotulinum neurotoxin serotype E neurotoxin heavy chain, a botulinumneurotoxin serotype F neurotoxin heavy chain and a botulinum neurotoxinserotype G neurotoxin heavy chain.
 6. The polypeptide of claim 1,wherein said botulinum neurotoxin light chain therapeutic element is abotulinum neurotoxin serotype A neurotoxin light chain, a botulinumneurotoxin serotype B neurotoxin light chain, a botulinum neurotoxinserotype C1 neurotoxin light chain, a botulinum neurotoxin serotype Dneurotoxin light chain, a botulinum neurotoxin serotype E neurotoxinlight chain, a botulinum neurotoxin serotype F neurotoxin light chainand a botulinum neurotoxin serotype G neurotoxin light chain.
 7. Acomposition comprising an active di-chain form of the cleavablesingle-chain polypeptide as defined in claim
 1. 8. A cleavablesingle-chain polypeptide comprising: a) a first amino acid sequenceregion comprising i) a first domain comprising a binding element peptideable to preferentially interact with a sensory afferent neuron cellsurface marker under physiological conditions; and ii) a second domaincomprising a botulinum neurotoxin heavy chain translocation element ableto facilitate the transfer of said single-chain polypeptide across avesicular membrane; and b) a second amino acid sequence regioncomprising a botulinum neurotoxin light chain therapeutic element havingSNARE-protein sequence-specific endopeptidase activity; c) a third aminoacid sequence region comprising a protease cleavage site, wherein theprotease cleavage site is cleaved by a non-human Enterokinase or atobacco etch virus protease; wherein said first and second amino acidsequence regions are separated by said third amino acid sequence region.9. The polypeptide of claim 8, wherein said polypeptide furthercomprises a fourth region comprising a target-binding portion of abinding tag.
 10. The polypeptide of claim 9, wherein said target-bindingportion comprises a His6, a monoclonal antibody, a maltose bindingprotein, a glutathione-S-transferase, a protein A, or a calmodulinbinding protein.
 11. The polypeptide of claim 8, wherein said botulinumneurotoxin heavy chain binding element is a botulinum neurotoxinserotype A neurotoxin heavy chain, a botulinum neurotoxin serotype Bneurotoxin heavy chain, a botulinum neurotoxin serotype C1 neurotoxinheavy chain, a botulinum neurotoxin serotype D neurotoxin heavy chain, abotulinum neurotoxin serotype E neurotoxin heavy chain, a botulinumneurotoxin serotype F neurotoxin heavy chain and a botulinum neurotoxinserotype G neurotoxin heavy chain.
 12. The polypeptide of claim 8,wherein said botulinum neurotoxin heavy chain translocation element is abotulinum neurotoxin serotype A neurotoxin heavy chain, a botulinumneurotoxin serotype B neurotoxin heavy chain, a botulinum neurotoxinserotype C1 neurotoxin heavy chain, a botulinum neurotoxin serotype Dneurotoxin heavy chain, a botulinum neurotoxin serotype E neurotoxinheavy chain, a botulinum neurotoxin serotype F neurotoxin heavy chainand a botulinum neurotoxin serotype G neurotoxin heavy chain.
 13. Thepolypeptide of claim 8, wherein said botulinum neurotoxin light chaintherapeutic element is a botulinum neurotoxin serotype A neurotoxinlight chain, a botulinum neurotoxin serotype B neurotoxin light chain, abotulinum neurotoxin serotype C1 neurotoxin light chain, a botulinumneurotoxin serotype D neurotoxin light chain, a botulinum neurotoxinserotype E neurotoxin light chain, a botulinum neurotoxin serotype Fneurotoxin light chain and a botulinum neurotoxin serotype G neurotoxinlight chain.
 14. A composition comprising an active di-chain form of thecleavable single-chain polypeptide as defined in claim 8.